Herpes Simplex Virus/Sleeping Beauty Vector-Based Embryonic Gene Transfer Using the HSB5 Mutant: Loss of Apparent Transposition Hyperactivity In Vivo

Abstract

The Sleeping Beauty (SB) transposon system has been successfully used as a gene delivery tool in nonviral and viral vector platforms. Since its initial reconstruction, a series of hyperactive mutants of SB have been generated. Questions remain as to whether the enhanced in vitro activities of these SB transposase mutants translate to the in vivo setting, and whether such increased integration efficiencies will ultimately compromise the safety profile of the transposon platform by raising the risk of genomic insertional mutagenesis. Herein, we compared the in vivo impact of a herpes simplex virus (HSV) amplicon-vectored ''wild-type'' SB transposase (SB10) and a ''hyperactive'' SB mutant (HSB5), codelivered in utero with the HSVT-bgeo transposable reporter amplicon vector to embryonic day 14.5 C57BL/6 mice. The SB10 and HSB5 transposases do not disparately affect the viability and development of injected mouse embryos. Quantitation of brain-resident bgeo expression on postnatal day 21 revealed that mice receiving HSB5 exhibited only a trending increase in transgene expression compared with the SB10-infused group, an outcome that did not mirror the marked enhancement of HSB5-mediated transposition observed in vitro. These findings indicate that in vivo application of hyperactive SB mutants, although not differentially genotoxic to the developing mouse embryo, does not necessarily provide a significant therapeutic advantage over the employment of a lesser active SB when delivered in the context of the HSV/SB amplicon platform.

Sleeping Beauty Transposition

“…The random genomic distribution of de novo SB insertions can be observed when the transposon DNA is introduced into the nucleus by extrachromosomal gene delivery, including plasmid vectors (114,115,116,117,119,122,125), integration-deficient lentiviral vectors (IDLVs) (114), adenovirus vectors (129), herpesvirus vectors (130), and adeno-associated vectors (131). In these cases, transposition takes place from the extrachromosomal vector into the genome.…”

Nonviral Gene Delivery with the Sleeping Beauty Transposon System

“…Retroviral vectors disabled in generating a cDNA copy of the retroviral vector have been shown to deliver the SB transposase mRNA into target cells with impressive efficiency (Galla et al, 2011). Last, herpes simplex virus vectors with a tropism toward infecting neural progenitor cells have been used to target SB insertions in the central nervous system in an in utero gene delivery system in the mouse (Bowers et al, 2006;de Silva et al, 2010). Furthermore, not only the procedures that are used to deliver the transposon vector components into cells, but those components themselves, may elicit unwanted effects in the cell.…”

Use of Transposon-Transposase Systems for Stable Genetic Modification of Embryonic Stem Cells

“…Such hybrid vectors certainly open new vistas in gene therapy, although rigorous testing should still be carried out to carefully examine the safety and efficiency of these methods. There is one study claiming that the chimera vector of a Herpes simplex virus and a hyperactive version of SB loses its transposition "hyperactivity" in vivo (de Silva et al, 2010a). However, as this study used an earlier version of the transposase, the results should be carefully repeated with the new SB100x system which provides a far more robust gene delivery in vivo than any previous transposons, therefore could potentially overcome this negative side effect.…”